Recombinant proteins are useful for a wide range of applications including, but not limited to, chemical and biological defense and the treatment and prevention of disease. Production of genetically engineered vaccine antigens, therapeutic proteins (including antibodies and antibody fragments), industrial enzymes, biopolymers, and bioremediation agents now constitute a multibillion dollar-per-year industry. There is also a large market for recombinant proteins in basic research (Pavlou and Reichert (2004); Langer (2005)).
Current platforms for the production of recombinant proteins are limited to a relatively small number of cell-based systems that include bacteria, fungi, and insect and mammalian tissue culture cells. Although bacteria can offer high yield and low cost alternatives for production of mammalian proteins, cell culture systems based on higher organisms (e.g., insect cells or mammalian cell systems) generally provide proteins having greater fidelity to the natural proteins in terms of protein folding and/or post-translational processing (e.g., glycosylation). Whole transgenic plants and animals have also been harnessed for the production of recombinant proteins, but the long development time from gene to final product can be a major drawback with these multicellular organisms, and purification of the recombinant proteins can be difficult and yield may be low.
Unicellular eukaryotes (e.g., Saccharomyces cerevisiae and Pichia pastoris) grow rapidly in inexpensive media and share some common pathways of protein folding, post-translational modification and protein targeting with more advanced organisms such as mammalian cells. Although the use of such unicellular eukaryotes for heterologous protein expression systems is known in the art, their rigid cell walls are an impediment to downstream protein purification.
After production of a desired recombinant protein within cells, the first step in isolating the protein is typically lysis of the cells. Lysis causes a forced mixing with the myriad of other cellular components, including proteases, which greatly complicates purification. In addition, lysis is problematic in expression systems that use microbial cells having rigid cell walls because the cell walls can impede downstream purification.
Although there are known methods, such as conventional chromatographic techniques (e.g., ion-exchange and affinity chromatography), for separating a desired protein from a mixture of proteins and/or cellular debris, such techniques can be inefficient and can require successive rounds of isolation over expensive column matrices to obtain highly purified products. These drawbacks add to manufacturing costs. Purification of recombinant proteins is a key factor in production costs, and even the most efficient systems consume between 25% and 80% of capital costs in the purification process (Frankel (2000)).
Most eukaryotic cells are capable of constitutive secretion. This is a process whereby proteins are delivered to the extracellular space via cargo vesicles that traffic to the cell surface by way of the endoplasmic reticulum (ER) and Golgi (Burgess and Kelly (1987)). This pathway has been harnessed for the production of recombinant gene products in a variety of systems and has significant advantages for protein purification because the process of secretion separates proteins of interest from the bulk of contaminating cellular material and obviates the need for cell lysis. Nonetheless, constitutive secretion has drawbacks as well. Typically, the process is slow and requires days to weeks to generate sufficient yields of a recombinant polypeptide for commercial use. In addition, thermal denaturation and the presence of proteolytic enzymes released into the culture medium can adversely affect the uniformity and function of the final protein product.
While most cells (including eukaryotic microbes) secrete proteins constitutively, there are some specialized cells that also store proteins in cortical secretory organelles (granules), which they discharge in a stimulus-dependent or regulated fashion (Burgess and Kelly (1987); Miller and Moore (1990); Gundelfinger et al. (2003)). In contrast with constitutive secretion, regulated secretion is triggered by the presence of chemical mediators known as secretagogues. Such mediators cause increased levels of intracellular calcium (Ca++) which, in turn, trigger fusion of cortical granules with the plasma membrane and release of the granules contents into the surrounding extracellular space. Depending on the level of the stimulus, regulated secretion can be an all or none phenomenon. In some cases, relatively large amounts of protein can be released within a period on the order of milliseconds. The principal advantage of regulated secretion is that recombinant proteins can be harvested rapidly, thus speeding the manufacturing process, and improving the quality of the final product, particularly when long incubation times have deleterious effects on protein function.
Stimulus-dependent secretion has been intensively studied in specialized mammalian cells such as neurons, β-cells of the pancreas, and mast cells, and methods for the production of recombinant proteins that rely on regulated secretion have been described in the prior art (e.g., U.S. Pat. Nos. 6,087,129; 6,110,707; 6,194,176; Grampp et al. (1992); Chen et al. (1995); Yang and Hsieh (2001)). These methods are drawn to the use of mammalian cells, and require that the gene for a protein that normally occupies the secretory granules (for example, insulin) be deleted and replaced by a gene for the recombinant protein (for example, prolactin) engineered to traffic to the same organelles. In all cases, the released proteins must be purified from culture supernatants using conventional chromatographic techniques following the addition of secretagogues to the growth media.
The use of mammal cells for the preparation of recombinant polypeptides can be further complicated by high costs and safety issues arising from the risks of mycoplasma or viral infections of the cell lines.
Therefore, there remains a need in the art for improved methods for rapid, high-fidelity and cost-effective production and purification of recombinant polypeptides.